RT-PCR primer design - Intron/exon boundaries - (May/08/2009 )

I have designed many primers (for old-school reverse transcriptase PCR, non of your newfangled real time here I'm afraid) and they have always worked fine however, I always have a niggling doubt about the whole intron/exon boundary thing. In the past I have designed my primers and then check the sequence in BLAT to check the sequence spans intron/exon boundaries. This always seems like a lot of work, is it really necessary? Sometimes it is very difficult to design primers that do this.

I hope this makes sense and someone can put my mind at rest over the weekend

Hellie on May 8 2009, 12:35 PM said:

On NCBI, you can search for your gene of interest. That cDNA sequence is the exons so you don't have to worry about intron boundaries. Then go to the blast section and at the bottom go to Primerblast, then blast your gene of interest to all other cDNA sequences and you;ll get a list of specific set ofprimers for your target of interest.

Thanks. That is what I have thought myself, I am just confused as I have read in various places that you should design primers to include more than one exon as to ensure that your product does not include genomic DNA. Am I making it more complicated that it needs to be?

The purpose of designing primers that bridge introns is to tell whether your RT-PCR product contains that from genome DNA, it is not for making sure that the primers are specific for your target sequence. For regular RT-PCR, I think it is a good practice to enforce this. Even for real-time PCR, although you don't usually run gels, it is still necessary to enforce it, otherwise any DNA contamination from RNA prep will appear as the same signal from cDNA.

pcrman on May 11 2009, 07:51 AM said:

That was my understanding of the reason for doing it, if there is genomic DNA in the PCR product then you would get a much larger product than expected. I was just confused because not every primer design protocol says you should do that so I wasn't sure if it was absolutely necessary. Is there a better way of searching for the intron boundaries other than manually looking through?

I believe it is necessary. As you have done, BLAT is a convenient tool for checking intron structure. Have you used in silico PCR also from UCSC genome broswer? Here is another one called Primer check at http://www.tigerteamconsulting.com/SpliceCenter/PrimerCheck

pcrman on May 11 2009, 09:19 AM said:

I have seen the insilico one but not primercheck. I will give that a try too. Thanks for your help, I am glad I was doing the right thing and not wasting time!